Preparation,Pharmacokinetics And Pharmacodynamics Evaluation Of Four Dihydroartemisinin Prodrug Self-assembled Nanoparticles

Objective:In order to improve the circulation time of dihydroartemisinin（DHA）in vivo,to achieve better antimalarial and antitumor effects,DHA prodrugs were synthesized by conjugating DHA and fatty amine（tetradecylamine,octadecylamine）with a disulfide bond（-SS-）or carbon-carbon bond（-CC-）.DHA prodrugs self-assembled nanoparticles(C₁₄-SS-DHA NPs,C₁₄-CC-DHA NPs,C₁₈-SS-DHA NPs,C₁₈-CC-DHA NPs)were prepared by nanoprecipitation method.The formulation properties,pharmacokinetics in rats,antimalarial activities in Plamodium yoelii rodent malaria,inhibition on 4T1 cells proliferation,and antitumor activity to the 4T1 tumor-bearing mice of DHA prodrugs self-assembled nanoparticles were evaluated,which provided basis and guidance for the treatment of malaria and anti-tumor applications for small molecule dihydroartemisinin prodrug self-assembled nanoparticles.Methods:1.Synthesis and characterization of DHA prodrugsDHA prodrugs were synthesized by esterification reaction and amidation reaction using DHA,ART,3,3’-Dithiodipropionic acid,tetradecylamine,octadecylamine.The structures of offinal products were identified by high resolution mass spectrometry（HR-MS）,and hydrogen spectroscopy（1H-NMR）.2.Preparation and characterization of DHA prodrugs self-assembled nanoparticlesDHA prodrugs self-assembled nanoparticles were prepared by nanoprecipitation method.The particle size and PDI were used as indicators to optimize single factor of formulation,and the preliminary stability of the prepared DHA prodrugs self-assembled nanoparticles were investigated.The entrapment efficiency and drug loading of DHA prodrugs self-assembled nanoparticles was determined in post-column derivatization method.In vitro release behavior of self-assembled nanoparticles of DHA prodrugs was evaluated by small cup method.3.Pharmacokinetics study of the DHA prodrugs self-assembled nanoparticles in ratsAfter the tail intravenous injection of DHA solution,C₁₄-SS-DHA NPs,C₁₄-CC-DHA NPs,C₁₈-SS-DHA NPs and C₁₈-CC-DHA NPs,blood samples were collected at determined time points.The DHA and DHA prodrugs of rat plasma were extracted by liquid-liquid extraction.Liquid chromatography-tandem mass spectrometry（LC-MS/MS）was used to study the pharmacokinetic characteristics of the DHA solution and DHA prdrugs self-assembled nanoparticles in rats.The DHA and DHA prodrugs were analyzed on a Kinetex?C8 column（50×2.1 mm,2.6μm;Phenomenex,USA）with a mobile phase consisting of acetonitrile and 10 mM ammonium acetate（v/v,90/10）at a flow rate of 300μl/min.Electro spray ionization source（ESI）in the positive ion mode was applied for analysis.Multiple reaction monitoring（MRM）at transitions m/z 302.3→163.2（DHA）,m/z 689.1→300.2(C₁₄-SS-DHA),m/z 717.2→328.2(C₁₆-SS-DHA),m/z 745.2→356.1(C₁₈-SS-DHA),m/z 597.1→314.3(C₁₄-CC-DHA),m/z 625.1→342.4(C₁₆-CC-DHA),m/z 653.2→370.3(C₁₈-CC-DHA),m/z 300.1→151.1（Artemisinin ART IS）was used to determined the concentration of DHA and DHA prodrugs.The relative pharmacokinetic parameters were calculated by using the statistical differences analysis method.4.Antimalarial activities of the DHA prodrugs self-assembled nanoparticlesThe antimalarial activities were investigated of five doses of DHA solution and DHA prodrugs self-assembled nanoparticle solution,respectively,in Plamodium yoelii rodent malaria using the four day suppression test method.One day after withdrawal,Wright-Giemsa stained thin blood smears was performed to calculate the infection rate,inhibition rate,ED₅₀（50%effective dose）,and ED₉₀（90%effective dose）.After stopping the drug for seven days,the infection rate among different groups were calculated and compared.The average days of survival time was recorded.5.Cytotoxicity test and antitumor activities of the DHA prodrugs self-assembled nanoparticlesThe cytotoxicity of DHA solution and DHA prodrugs self-assembled nanoparticles were studied by MTT method using 4T1 cells as model cells.The cytotoxicity of the different formulations was evaluated by comparing with the IC₅₀ values.Balb/c mice inoculated with 4T1 cells subcutaneously were used as model animals.The antitumor activities of the mice were assessed after intravenous injection of DHA solution and DHA prodrugs self-assembled nanoparticle solutions.The antitumor activity of each preparation was evaluated by comparing the tumor growth curves,the weight changes and the tumor load of mice in different groups.Results:1.Synthesis and characterization of the DHA prodrugsThe DHA prodrugs were successfully synthesized identified by HR-MS and 1H-NMR.C₁₄-SS-DHA is a pale yellow viscous liquid with a yield of 58%;C₁₄-CC-DHA is a white solid,yield 75%,C₁₈-SS-DHA is a pale yellow waxy solid,yield 61%;C₁₈-CC-DHA is a white waxy solid with a yield of 77%2.Preparation and characterization of DHA prodrugs self-assembled nanoparticlesThe DHA prodrugs self-assembled nanoparticles were prepared by nanoprecipitation,which showed a spherical shape and well-proportioned distribution in particle size.Different DHA prodrugs self-assembled nanoparticle solutions were prepared after a single factor optimization.The particle size was between 100¹40 nm,the PDI was less than 0.3,and the absolute value of the zeta potential was between 20-45 mV.The encapsulation efficiency of DHA prodrugs self-assembled nanoparticles were between 93%⁹7%,and the drug loading of DHA prodrugs self-assembled nanoparticles were between 79%⁸0%.The stability experiment indicated that different kinds of prodrugs self-assembled nanoparticles could be stably stored at 4°C for one month.The cumulative release of prodrugs in C₁₄-SS-DHA NPs,C₁₄-CC-DHA NPs,C₁₈-SS-DHA NPs,and C₁₈-CC-DHA NPs reached 80.3%,79.2%,72.9%and 71.2%,respectively,within 6 hours.In the presence of dithiothreitol,the cumulative release of DHA in C₁₄-SS-DHA NPs and C₁₈-SS-DHA NPs reached 40%and 62%,respectively,within 60 hours.3.Pharmacokinetics study of the DHA prodrugs self-assembled nanoparticles in ratsAfter intravenous injection of DHA solution,C₁₄-SS-DHA NPs,C₁₄-CC-DHA NPs,C₁₈-SS-DHA NPs and C₁₈-CC-DHA NPs to rats,the area under the plasma concentration curve of DHA in plasma(AUC_0-t)was 908.9±82.3,23211.5±9101.9,4221.5±540.0,748.7±221.7 and 13212.2±1765.1 h·μg·L^-1,respectively.The order of AUC_0-t is C₁₄-SS-DHA NPs>C₁₈-CC-DHA NPs>C₁₄-CC-DHA NPs>DHA solution>C₁₈-SS-DHA NPs.MRT were 0.3±0.1,0.9±0.3,0.2±0.1,1.7±0.3 and 1.6±0.3 h,respectively.The order of MRT is C₁₈-SS-DHA NPs≈C₁₈-CC-DHA NPs>C₁₄-SS-DHA NPs>DHA solution≈C₁₄-CC-DHA NPs.The concentration of C₁₄-CC-DHA in plama can not be detected when C₁₄-SS-DHA NPs injected into rats.The AUC_0-t（DHA prodrug in plama）of C₁₄-SS-DHA NPs,C₁₈-SS-DHA NPs and C₁₈-CC-DHA NPs in rat plasma were24179.2±1885.5,92525.3±25760.4 and 64123.3±14088.2 h·μg·L^-1;MRT were 0.8±0.1,1.1±0.3 and 1.3±0.4 h,respectively.4.Antimalarial activities of the DHA prodrugs self-assembled nanoparticlesAfter withdrawal for one day,the ED₅₀ values of C₁₄-SS-DHA NPs,C₁₄-CC-DHA NPs,C₁₈-SS-DHA NPs and C₁₈-CC-DHA NPs were measured.The ED₅₀0 values of those groups were 0.14±0.05,0.20±0.09,0.03±0.01 and 0.04±0.01μmol/kg,respectively,which were significantly lower that of DHA group（0.87±0.27μmol/kg）（P<0.05）.After one week of withdrawal,the infection rate of parasites in each group increased,but the infection rate of the DHA prodrugs self-assembled nanoparticles was still lower than that of DHA solution group.The average survival time of the DHA prodrugs self-assembled nanoparticles in each dose was longer than that of the DHA solution group.The infection rate of plasmodium gradually reduced in the same formulation administration group with increase of dose,in a dose-dependent manner.5.Cytotoxicity test and antitumor activities of the DHA prodrugs self-assembled nanoparticlesThe additive inhibition to 4T1 cells in the DHA solution group and different kinds of the DHA prodrugs self-assembled nanoparticles were enhanced with the increase of drug concentration and incubation time,which had dose and time dependence.were incubation with cells for 48h.when these preparation were incubated with cells for 48h,the IC₅₀values of DHA solution,C₁₄-SS-DHA NPs,C₁₄-CC-DHA NPs,C₁₈-SS-DHA NPs and C₁₈-CC-DHA NPs were 2.60±0.22,2.05±0.05,2.52±0.20,3.43±0.10 and 3.86±0.10μmol/L,respectively.The results of tumor growth curve showed that the tumor growth rate in the DHA solution,C₁₄-SS-DHA NPs,C₁₄-CC-DHA NPs,C₁₈-SS-DHA NPs and C₁₈-CC-DHA NPs groups were slower than that in the saline group.The tumor burden of the saline group,DHA solution,C₁₄-SS-DHA NPs,C₁₄-CC-DHA NPs,C₁₈-SS-DHA NPs and C₁₈-CC-DHA NPs were（13.4±2.2）%,（7.8±0.67）%,（5.5±1.3）%,（9.4±0.5）%,（7.2±0.2）%and（5.9±0.6）%,respectively.Conclusion:1.Four kinds of DHA prodrugs were successfully synthesized by conjugating DHA and fatty amine（tetradecylamine,octadecylamine）with disulfide bonds or carbon-carbon bonds as link.2.The DHA prodrugs self-assembled nanoparticles were prepared using a sample mothed,which had spherical shape,small particle size,high encapsulation efficiency and drug loading capacity,and could be stably stored at 4°C for one month,in the presence of dithiothreitol,DHA in C₁₄-SS-DHA NPs and C₁₈-SS-DHA NPs have a faster release.3.After intravenous administration,the DHA prodrugs self-assembled nanoparticles prolonged the biological half-life of DHA to varying degrees and improved the AUC_0-t-t compared with DHA solution.DHA prodrugs self-assembled nanoparticles might have a positive effect on the pharmacodynamics of this class of drugs.4.After the tail vein injection of malaria mice,the antimalarial effect gradually increased with the increase of the dose.The antimalarial activities of the DHA prodrugs self-assembled nanoparticles were stronger than that of DHA solution（P<0.05）.The same kind of tethered arm prodrugs increased the antimalarial activity with the DHA prodrugs of octadecylamine as carrier.The DHA prodrugs self-assembled nanoparticles with the same kind of carrier containing the disulfide bond had stronger antimalarial activity than the prodrugs self-assembled nanoparticles with the carbon-carbon bond as the linking arm.5.The cytotoxicity of different DHA prodrugs self-assembled nanoparticle on 4T1 cells increased with the increase of dose and incubation time.The DHA prodrugs of the same kind of tether,the tetradecylamine-modified DHA prodrugs self-assembled nanoparticles have better inhibition than the octadecylamine-modified DHA prodrugs self-assembled nanoparticles.The DHA prodrugs of the same kind of fatty amine as the carrier,and the cytotoxicity of the DHA prodrugs self-assembled nanoparticle containing the disulfide bond was stronger than the carbon-carbon bond.The DHA prodrugs self-assembled nanoparticles had a certain antitumor effect on 4T1tumor-bearing mice,and C₁₄-SS-DHA NPs and C₁₈-CC-DHA NPs have the best anti-tumor effect.